title: Advanced oxidation protein products induce inflammatory response in fibroblast-like synoviocytes through NADPH oxidase -dependent activation of NF-κB.
abstract: BACKGROUND
  Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro.
  
  
  METHODS
  FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin(IL)-1β, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB).
  
  
  RESULTS
  Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-α, IL-1β, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47(phox), triggered an interaction between p47(phox), p22(phox) and gp91(phox), and significantly upregulated expression of NADPH oxidase subunits p47(phox), p22(phox) and gp91(phox). IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin.
  
  
  CONCLUSIONS
  These data indicate that AOPPs induce inflammatory response in FLSs is medicated through NADPH oxidase-dependent activation of NF-κB.
authors.affiliation: Department of Orthopedic Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Zheng
authors.forename: Zhao-Ming
authors.initials: ZM
authors.lastname: Zhong
authors.forename: Shuai
authors.initials: S
authors.lastname: Qin
authors.forename: Guo-Xian
authors.initials: GX
authors.lastname: Chen
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.forename: Ji-Huan
authors.initials: JH
authors.lastname: Zeng
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Ye
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Kai
authors.initials: K
authors.lastname: Yuan
authors.forename: Ling
authors.initials: L
authors.lastname: Yao
authors.forename: Jian-Ting
authors.initials: JT
authors.lastname: Chen
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D062107:Advanced Oxidation Protein Products / Q000494:pharmacology*; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007249:Inflammation / Q000139:chemically induced* / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013482:Superoxide Dismutase / Q000378:metabolism; D013583:Synovial Membrane / Q000166:cytology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D062107:Advanced Oxidation Protein Products; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D000111:Acetylcysteine